A technology of producing saccharides from a lignocellulose raw material that has been subjected to a treatment appropriate for saccharification, is a technology beneficial to the formation of a recycling-oriented society since alcohols that may be used as a gasoline substitute fuel, or chemical raw materials such as succinic acid and lactic acid that may be used as raw materials for plastics can be produced by using the saccharides as the fermentation substrates for microorganisms.
Methods for producing monosaccharides or oligosaccharides that may be used as fermentation substrates from the polysaccharides in a plant-based biomass, can be broadly classified into two types. One type is an acid saccharification method of hydrolyzing polysaccharides using a mineral acid, and the other type is an enzymatic saccharification method of hydrolyzing polysaccharides using an enzyme or a microorganism which produces the enzyme.
The acid saccharification method is technically complete as compared with the enzymatic saccharification method. However, in the case of the method of using a lignocellulose-based biomass as a raw material, the saccharide yield is low as compared with the method of using starch or molasses as a raw material, and also, the fact that a facility for treating waste acids discharged from treatment processes, or a large-sized facility capable of enduring the corrosion caused by acid is needed, causes an increase in the product cost, so that these pose a serious problem for practical application.
On the other hand, in regard to the enzymatic saccharification method, due to the decrease in the price of enzymes in recent years and the progress of technology, the total cost including post-treatments is becoming closer to the cost of the acid saccharification method. However, because the price of enzymes which accounts for a high proportion of the total cost of the enzymatic saccharification method is still high, in order to achieve practical application of the enzymatic saccharification method, a further decrease in the cost of the enzymes is important.
As a technology for decreasing the cost of the enzymatic saccharification method, the development of a method for a pretreatment that facilitates the access of an enzyme to cellulose fibers, or the development of a method of efficiently saccharifying crystalline cellulose, and the development of a method for efficient recovery and reuse of enzymes can be considered.
A lignocellulose material from which lignin has not been removed is not easily degraded by enzymes as compared with a lignocellulose material from which lignin has been removed, and is not saccharified so that the lignocellulose material from which lignin has not been removed remains as a residue in the saccharification liquid together with impurities such as resins and metals. In general, this residue is separated by screening, centrifugation or the like and is discarded. Since this residue contains a large amount of adsorbed enzymes, which occupy a large proportion of the cost in the enzymatic saccharification method, there is a problem that if the residue separated from the reaction liquid is directly discarded, the highly expensive enzymes are also discarded. That is, it is desired to recover and effectively utilize the residue for the purpose of reducing the cost of the enzymatic saccharification method. In regard to the technology of reusing the residue recovered in the enzymatic saccharification method, there have been reports on a method of combusting the residue and obtaining heat energy (PTL 5), a method of subjecting the residue to hydrothermal gasification, and synthesizing ethanol from the produced synthesis gas using an ethanol synthesis catalyst (PTL 6), a method of utilizing the residue as a fuel or a fertilizer (PTL 7), and a method of utilizing the residue as heat energy (PTL 8). However, since these methods cause large increases in the cost as a result of the addition of treatment processes, in the case of designing a practically useful facility, it cannot be said that these methods are satisfactory as methods for addressing the problem of cost reduction.
As means for recovering enzymes in the residue such as described above, washing of the residue can be taken into consideration. However, since the enzymes are firmly bound to cellulose through the cellulose binding domain (CBD) that specifically adsorbs to cellulose, which is carried by the enzymes in the molecules, it has been difficult to sufficiently recover the enzymes that have been adsorbed to cellulose, by simple water washing.
Thus, for the purpose of improving the recovery ratio of enzymes, a method of treating the enzymes by adding a surfactant (see PTL 1), and the like have been suggested. However, even in the surfactant treatment method, it cannot be said that the recovery ratio of enzymes is satisfactory, and the method is not practical from the viewpoint that there is a concern for the deactivation of enzymes due to the addition of chemicals, an increase in the cost as a result of the addition of treatment processes, and the adverse effects on microorganisms in the fermentation step that follows.
As the method of recovering enzymes from a saccharide solution, a method of using ultrafiltration (see PTL 2), a method of adsorbing and recovering enzymes by adding cellulose again to the saccharide solution (see PTL 3), and the like have been suggested. However, the ultrafiltration method has a problem that fine impurities clog the filtration membrane, and thus a sufficient treatment speed and a sufficient enzyme recovery ratio cannot be obtained, and it is difficult to achieve sufficient enzyme recovery with the method of recovering by cellulose addition.
A method of reusing a lignocellulose residue to which enzymes are adsorbed, in the subsequent batch of enzymatic saccharification, without going through a step of detaching the adsorbed enzymes has been suggested (PTL 4).
In this method, since the accumulation of the residue cannot be avoided, there is a concern for a decrease in the reaction efficiency. Furthermore, in connection with enzymes having the CBD, such as CBH (cellobiohydrases), recycling of enzymes can be achieved by re-treating the lignocellulose residue in the subsequent batch; however, since there are occasions in which β-glucosidases and the like are liberated in the supernatant, it is difficult to recycle all of the cellulases that have been added.
Also, in this method, because the undegraded residue itself is in a state of being not easily degradable even if mixed again with an enzyme solution, it is desired to bring the undegraded residue to a state of being easily saccharifiable. The inventors of the present invention found that when an undegraded residue recovered by solid-liquid separation is mechanically treated and is subjected to saccharification and fermentation again, the ethanol output is increased (PTL 9). However, this method has a problem that the residue that has been used as a raw material is a residue recovered by using a 420-mesh (38-μm) screen and includes fibers of a wide range of sizes. Fibers of large sizes with a large amount of adsorbed lignin are not easily degraded by enzymes, and are therefore not sufficiently saccharified if not subjected to a pretreatment (mechanical treatment or the like). If only those fibers of small sizes with a small amount of adsorbed lignin could be selectively recovered and enzymatically saccharified again as a raw material without being subjected to a pretreatment, an enhancement of the ethanol output with high efficiency could be expected.
As a method of decreasing the cost of enzymes, methods of recycling enzymes have been reported. According to the method of Scott, C. D. and colleagues (Non-PTL 1), a continuous system is contemplated, which is provided with a recycle line having a grinding apparatus based on a high speed centrifugal pump which, in a main reaction tank which enzymatically hydrolyzes an old paper raw material by adding a large amount of an enzyme (80 to 160 units relative to 1 g of a substrate in terms of filter paper degradation activity), removes produced glucose and cellobiose components with high shear force on the surface of unreacted old paper in the enzymatic hydrolysate, and thereby always exposes new cellulose fiber surfaces; a membrane separation apparatus which separates the unreacted raw material and the hydrolysate from the treatment liquid coming from the grinding apparatus, and circulates only the unreacted raw material to the main reaction tank; and a filtering apparatus which separates enzymes and produced glucose and cellobiose from the hydrolysate coming from the membrane separation apparatus, and circulates only the enzymes to the main reaction tank, and the cost is predicted. According to this system, the saccharification ratio is 100% in 25 hours, and the residual ratio of enzymes is 95% or higher in 24 hours. Also, it is described that enzymes adsorb to the residue and are eliminated, that the adsorptive function of enzymes to the residue may be decreased by increasing the pH to 5 to 7, and it is reported that the adsorptive function of enzymes can be decreased by lowering the temperature to 5° C.
As a method of recovering and reusing the enzymes, a method has been reported in which birch wood that has been steam blasting treated is added to a saccharification tank at a concentration of 5%, 20,000 units of a cellulase is added thereto, a saccharide solution and an enzyme solution are separated by ultrafiltration, and while the enzyme is recovered and reused, 630 g of monosaccharides are obtained from 2 kg of birch wood over 8 days. It is considered that the amount of the enzyme used could be saved by 20% by this method (Non-PTL 2).